Effect on turkey spermatozoa of a frothy fluid derived from the cloaca of a male turkey

The influence on turkey spermatozoa of a frothy fluid derived from the cloacal region of a male turkey was investigated. The frothy fluid was collected from the turkey tom during mounting, and semen for the experiment was obtained from the ductus deferens removed after necropsy. Spermatozoa diluted with frothy fluid were examined for motility, viability, and fertilizing capacity and compared with semen diluted with phosphate buffer or undiluted ductal semen. The life-span of spermatozoa suspended in frothy fluid was slightly prolonged during in vitro storage as compared with the undiluted semen or the semen diluted with phosphate buffer; however, a rapid increase of the number of deformed spermatozoa during storage was observed in the semen diluted with frothy fluid. The fertilizing ability of spermatozoa was not influenced by the dilution with frothy fluid when the diluted spermatozoa were inseminated intravaginally immediately after the dilution. On the contrary, when spermatozoa suspended in frothy fluid were preserved at 0 C for 24 h, their fertilizing capacity decreased drastically, probably due to the increased number of abnormal spermatozoa during in vitro preservation.     

Effects of hypothermic storage on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and plasma membrane integrity in striped bass (Morone saxatilis) sperm

Experiments were conducted to determine the effect of hypothermic 24 h storage on striped bass sperm cell plasma membrane integrity, free intracellular Ca²⁺ ([Ca²⁺]_i), mitochondrial membrane potential (ΔΨ_m), and reactive oxygen species (ROS) formation (oxidation of hydroethidine to ethidium) as determined by flow cytometry; motion activation and ATP concentration as determined by Luciferin-Luciferase bioluminescence assay. Semen was stored for 1 or 24 h at 4 °C in an O₂ atmosphere undiluted or diluted (one volume semen with 3 volumes diluent) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH 8) or with seminal plasma in the presence of various treatments. Viability (% cells excluding propidium iodide) approached 100% after 1 h storage in undiluted or diluted semen. After 1 h of storage the [Ca²⁺]_i marker, Fluo-3, was detected in only 3% of sperm cells in undiluted or diluted semen. In contrast to storage for 1 h, after 24 h the incidence Fluo-3 fluorescence intensity was increased (P < 0.05) in > 50% of the viable cells in undiluted and diluted semen along with increased cell death; the presence of 1 mM ethylene glycol tetraacetic acid (EGTA) blocked CaCl₂-induced Fluo-3 fluorescence and cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased (P < 0.05) to 30% after 24 h. However, motility activation failed in the presence of EGTA at 1 or 24 h. During storage ΔΨ_m was not affected by storage time or treatment. In contrast, sperm ATP was greater (P < 0.05) at 1 h than at 24 h and was greater in sperm stored in diluted than undiluted form. While ROS formation was induced by menadione treatment, there was no evidence of storage-induced ROS formation in the absence of menadione. The increased [Ca²⁺]_i found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.     

Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fractions was combined with 50% of the original volume of SP fractions for each ejaculate to represent total ejaculates. SR and total ejaculates were diluted with skim milk-glucose semen extender as follows: 1) no dilution, or dilution to 2) 100 x 10(6)sperm/ml, 3) 50 x 10(6)sperm/ml, or 4) 25 x 10(6)sperm/ml. Semen samples were evaluated at 0.5, 3, 6, 12, and 24 h postejaculation (25 degrees C storage temperature) for percentages of total spermatozoal motility (TSM) and progressive spermatozoal motility (PSM). Mean TSM was greater (P < 0.05) in SR ejaculates than total ejaculates at 12 and 24 h postejaculation. Mean TSM of undiluted semen was lower (P < 0.05) than other dilution ratios over all periods. Mean TSM was greater (P < 0.05) at a 25 x 10(6)sperm/ml dilution ratio than a 50 x 10(6)sperm/ml dilution ratio at 12 and 24 h postejaculation, and greater (P < 0.05) than a 100 x 10(6)sperm/ml dilution ratio from 3 to 24 h postejaculation. Similar patterns were found for PSM. Collection of SR ejaculates and dilution to 25 x 10(6)sperm/ml improved longevity of spermatozoal motility.     

Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 degrees C

Artificial insemination (AI) in rabbits is not extensive in the breeding programs of the rabbit meat industry. A limiting factor is related to the semen preservation. In order to improve the use of AI, two experiments have been conducted to evaluate sperm viability and fertility of rabbit semen chilled and stored at 15 degrees C after dilution in Tris-based extenders. In Experiment 1, pooled semen samples were diluted 1:10 (semen/extender) in four different Tris-based extenders (Tris-citric-glucose (TCG), TES-Tris-glucose (TTG), Tris-citric-fructose (TCF) and TES-Tris-fructose (TTF)) and stored at 15 degrees C. Sperm viability was evaluated at 0, 24, 48, 72 and 96 h following dilution for total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (NAR). Viability of spermatozoa declined with time of storage (P<0.05), irrespective of the extender used. There were interactions between extender and time of storage (P<0.05) in all viability parameters evaluated. After 96 h of storage, TCG provided the highest sperm viability (P<0.05) and TTG the lowest (P>0.05). In Experiment 2, a field trial was conducted at a commercial farm to evaluate the conception and farrowing rates of rabbit spermatozoa extended in TCG. After synchronization of oestrous and induction of ovulation, 3713 does with different physiological conditions (nulliparous, primiparous, lactating and re-breeding) were inseminated one time (15x10(6) sperm per doses) with semen stored at 0 (n: 1275), 24 (n: 1503) and 48 h (n: 935) at 15 degrees C. Overall conception and farrowing rates were 77.1+/-0.7 and 70.4+/-0.7, respectively, and the mean litter size was 7.6+/-0.1. Fertility results were unaffected by the time of semen storage (P>0.05). Regardless of time of semen storage, fertility results were affected by the physiological conditions of does (P<0.05). Nulliparous and lactating does showed the highest fertility and primiparous the lowest. In summary, these results indicate that Tris-buffer extenders are effective for preserving viability and fertilizing capability of rabbit spermatozoa stored at 15 degrees C.     

Influence of antibiotics on bacterial load and sperm parameters during short-term preservation of collared peccary semen

Studies on semen and sperm cells are critical to develop assisted reproductive technologies for the conservation of the collared peccary. The objective of the study was to compare the effect of different antibiotics on the bacterial load and sperm quality during short-term storage of peccary semen. Fresh semen samples from 10 males were extended in Tris-egg yolk or Tris-Aloe vera supplemented with streptomycin-penicillin (SP; 1 mg/mL - 1000 IU/mL or 2 mg/mL - 2000 IU/mL) or gentamicin (30 µg/mL or 70 µg/mL) before storage at 5°C. Bacterial load and sperm motility, membrane integrity and function, mitochondrial activity, and morphology, were evaluated at different time points for 36 h. The SP and gentamicin treatments concentration inhibited (p < 0.05) bacterial growth for 36 h regardless of the extender. Compared to the other treatments, Tris-egg yolk plus 70 µg/mL gentamicin maintained the sperm parameters for longer, including total motility (41.9 ± 6.1%) at 24 h, and membrane integrity (58.3 ± 2.1%) at 36 h. In contrast, the highest SP concentration in both extenders impaired sperm membrane integrity at 36 h (p < 0.05). For the liquid storage of collared peccary semen, it therefore is recommended to use Tris extender supplemented with egg yolk and gentamicin (70 µg/mL).     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

Seminal plasma addition attenuates the dilution effect in bovine sperm

Dilution of semen to low cell numbers/dose can result in a bull-dependent reduction in the post-thaw viability of cryopreserved bovine spermatozoa. It is possible that essential seminal plasma components are lacking at the greater dilution rates, thereby contributing to the deleterious effects of semen dilution. Ejaculates of 6 Holstein bulls were diluted to 120 x 10(6) sperm/mL in an egg yolk citrate extender (EYC). Split samples were further diluted to 80, 40, 20 and 4 x 10(6) sperm/mL in EYC extender with (+SP) and without (-SP) the addition of frozen/thawed seminal plasma previously obtained from a vasectomized bull. Serial dilutions for the +SP treatments were calculated and performed such that each dilution contained a volume of seminal plasma equal to the original 120 x 10(6) sperm/mL dilution. Samples were then loaded into 0.5-mL French straws yielding final sperm concentrations of 30, 20, 10, 5 and 1 x 10(6)/dose. Straws from each dilution were analyzed using 2 stain combinations: the sperm viability stain, SYBR-14 and propidium iodide (PI); or the mitochondrial-specific, membrane potential-dependent stain JC-1 along with PI. Split-plot analysis of variance indicated that within bulls, there were greater proportions of viable spermatozoa in aliquots containing added seminal plasma than in aliquots without added seminal plasma (P < 0.05). Contrast analyses showed that sperm viability significantly decreased as sperm concentration decreased in the -SP samples. Although the dilution effect was also observed in the +SP samples, the magnitude of the effect was less than in the -SP samples. At most sperm concentrations, the proportions of spermatozoa that stained with JC-1 were correlated (r > 0.84; P < 0.05) with the percentages of SYBR- 14 stained spermatozoa. Furthermore, the proportions of JC-1-stained spermatozoa were greater in the +SP aliquots than in the -SP samples at a concentration of 10 x 10(6) sperm/0.5 mL. These results suggest that the addition of seminal plasma can be beneficial to sperm viability when semen is diluted to low cell numbers/dose.     

Improvement of rooster semen quality using coenzyme Q10 during cooling storage in the Lake extender

Sensitivity of rooster semen to stressful condition of cooling restricts the semen storage in commercial flocks for artificial insemination. This study was accomplished to investigate the effect of coenzyme Q10 (CoQ10) addition to the Lake extender during chilled-storage on the parameters of sperm quality and fertility performance. Roosters’ pooled semen samples were assigned into equal parts and diluted with Lake extender supplemented with different concentrations of CoQ10 (0, 1, 2, 5 and 10 μM CoQ10). Then, semen samples were cooled to 5 °C and stored over 48 h. Total and progressive motilities, abnormal morphology, viability, membrane functionality, lipid peroxidation (LPO) and mitochondria active potential of diluted sperm were evaluated at 0, 24 and 48 h of cooling storage. Fertility performance of cooled stored semen was examined at 24 h of cooling storage. Although CoQ10 did not affect sperm quality at the starting time of cooling storage (0 h), extender supplementation with 5 μM of CoQ10 showed higher (P ≤ 0.05) sperm total and progressive motilities, membrane functionality, viability and mitochondria active potential at 24 h as well as total motility, viability and membrane functionality at 48 h in contrast with other groups. Moreover, lipid peroxidation was lower (P ≤ 0.05) in semen samples diluted with 5 μM CoQ10 at 24 and 48 h compared to others. After artificial insemination with 24 h chilled-stored sperm, fertility efficiency was higher (P ≤ 0.05) in treatments contained 5 μM CoQ10 compared to the control group. According to the results, using optimum dose of CoQ10 could be helpful to save rooster semen against chilled storage structural and functional damages.     

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.     

Effect of antioxidants on preservation of motility,viability and acrosomal integrity of equine spermatozoa during storage at 5 degrees C

Preservation of liquid semen at 5 degrees C is an important technique in the breeding management of horses. Oxidative damage to spermatozoa during storage is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. The objective of this study was to evaluate the use of water-soluble and lipid-soluble antioxidants to improve the maintenance of motility of equine spermatozoa at 5 degrees C during storage for 72 to 96 h. In Experiment 1, the effect of addition of catalase on the maintenance of motility, viability and acrosomal integrity was determined. Semen was collected, and these treatments were applied: catalase (0, 100 or 200 U/mL) in nonfat, dried skim milk extender (NFDSM; with or without seminal plasma) or 10% seminal plasma + NFDSM. Motility was determined by computerized semen analysis (CASA) at 0, 24, 48 and 72 h. Viability and acrosomal integrity were determined at 72 h of storage. There was no significant treatment effect on the maintenance of sperm motility during 72 h storage. In Experiment 2, the effect of adding lipid-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted to a final concentration of 25 x 10(6) sperm/mL in NFDSM containing butylated hydroxytoluene (BHT; 2.0, 1.0, or 0.5 mM), Vitamin E (4.0, 2.0, 1.0 mM), or Tempo (2.0, 1.0, or 0.5 mM). Although the addition of BHT significantly reduced (P < 0.05) progressive motility during storage compared to the control, there were no positive treatment effects of either Vitamin E or Tempo on maintenance of motility. In Experiment 3, the effect of adding water-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted in NFDSM containing these treatments: Trolox (2.0 mM), Tempo (1.0 mM), Vitamin C (0.45 mg/mL), BSA (3% w/v), combinations of these antioxidants, or control. Adding these water-soluble antioxidants did not significantly improve the maintenance of motility during cooled storage at 5 degrees C. In conclusion, adding the enzyme scavenger, catalase, or a variety of lipid- and water-soluble antioxidants did not significantly improve the maintenance of motility during liquid semen storage at 5 degrees C.     

Fertility of undiluted ram epididymal spermatozoa stored for several days at 4°C

In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.     

The preservability of turkey semen quality during liquid storage in relation to strain and age of males

It is difficult to maintain turkey semen quality after in vitro liquid storage and the problem is worsened by animal aging. Little is currently known about the effects of both reproductive period and strain on the preservability of qualitative characteristics of turkey semen during liquid storage. The purpose of this study was to evaluate the effect of the reproductive period of two commercial turkey strains on semen quality changes during in vitro storage for upto 48 h at 5 degrees C. Two different periods were considered: first period from 32 to 40 weeks of age and the second one from 44 to 52 weeks. Turkey males from either British United Turkeys (BUT) Big-6 line and Hybrid Large White line (Hybrid) were used. Semen pools of each tom strain were diluted with Beltsville Poultry Semen Extender (BPSE) and the motility, viability and membrane integrity of sperm were evaluated at 3, 24 and 48 h of liquid storage at 5 degrees C. The sperm concentration was significantly affected by period (P<0.01) and strain (P<0.05), with best values in first period and in the Hybrid semen. Besides also the motility, viability and membrane integrity during 48 h of storage were better (P<0.05) in the first period compared to the second one for both strains, particularly in Hybrid semen. During storage it was clearly shown in the first period that Hybrid sperm worsened more than the BUT one: in spite of the motility and viability values were at first (3h) higher (P<0.05) in Hybrid semen, after 48 h of storage the motility did not show any significant difference between strains while the viability resulted even better (P<0.05) in BUT semen. In the second period, although the semen quality decreased during the storage with a similar trend for both strains, better (P<0.05) values were found in BUT semen. Our results indicated that the reproductive period affected the quality of turkey semen in a different manner according to the strain. Moreover BUT semen showed a better in vitro storage ability compared to the Hybrid one.     

Milk caseins decrease the binding of the major bovine seminal plasma proteins to sperm and prevent lipid loss from the sperm membrane during sperm storage

Milk is used as a medium for sperm preservation. Caseins, the major proteins of milk, appear to be responsible for the protective effect of milk on sperm. Recently, we have shown that egg yolk, which is also widely used to preserve semen, protects sperm functions by preventing the binding to sperm of the major proteins of bull seminal plasma (BSP proteins), thereby preventing BSP protein-mediated stimulation of lipid loss from the sperm membrane. In the present study, we investigated whether milk caseins protect sperm in the same manner as egg yolk. Bovine ejaculates were diluted with skimmed milk permeate (skimmed milk devoid of caseins) or permeate that was supplemented with caseins and stored at 4 degrees C for 4 h. In the semen diluted with permeate, sperm viability and motility decreased in a time-dependent manner. However, in semen diluted with milk or permeate supplemented with caseins, sperm functions were maintained. In addition, lower amounts of the BSP proteins were associated with sperm in semen diluted with milk or permeate supplemented with caseins, as compared to semen diluted with permeate. No milk proteins were detected in the sperm protein extracts. Furthermore, sperm diluted with milk or permeate supplemented with caseins showed 3-fold lower losses of cholesterol and choline phospholipids than sperm diluted with permeate during storage. Thus, milk caseins decreased the binding of BSP proteins to sperm and reduced sperm lipid loss, while maintaining sperm motility and viability during storage. These results support our view that milk caseins prevent the detrimental effects of BSP proteins on the sperm membrane during sperm preservation.     

Chilled storage of semen from Atlantic halibut, Hippoglossus hippoglossus L. II: effect of spermiation advancement, catheterization of semen, and production-scale application

A method for in vitro storage of Atlantic halibut Hippoglossus hippoglossus L. semen can facilitate seed production. The study aimed at determining the effect of male spermiation advancement on viability of chilled stored spermatozoa. The use of catheterization of semen from the sperm duct was examined. Also, large volumes of semen were stored under sub-optimal production-like conditions in order to determine the suitability of the method into practical use. Semen was collected from two broodstocks: natural photoperiod males, being at the first phase of the reproductive season and 3-month advanced photoperiod males, being at the end of the reproductive season. Semen samples were diluted 1:5 (v/v) with modified Hanks' Balanced Salt Solution supplemented with antibiotics, and stored in Ziploc bags filled with air. Sperm motility parameters, assessed by computer-assisted sperm analysis (CASA), were assessed weekly. Experimental and production-scale fertilization trials were performed. Sperm samples from natural photoperiod males showed significantly longer viability under in vitro storage conditions than sperm from advanced photoperiod males. In the natural photoperiod group, the decrease in spermatozoa motility, curvilinear velocity and straight-line velocity occurred on day 50, 14, and 28 of storage, respectively. Spermatozoa from one of five males were still motile on day 80 of storage, and fertilization rates and embryo survival rates obtained using semen stored for 70 days did not differ from control values and they were significantly higher than values obtained with the use of fresh semen of the same male, but being at the end of reproductive season. Catheterization of semen showed no advantage to stripping the semen without a catheter, even for samples stored undiluted for 1 day of collection, before dilution. Under sub-optimal conditions, spermatozoa stored in large volumes (10-100mL of diluted semen) without any special treatments except for weekly swirling, remained viable for more than 1 month. Production-scale fertilizations with samples stored for 5-21 days resulted in high survivals of embryos and hatchlings. Because of its simplicity and efficiency, the method shows a high potential for use in commercial Atlantic halibut farming. It has already been applied to a halibut breeding programme for the next reproductive season at our research station.     

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird’s Nest on the quality of chilled Arabian stallion semen

The aim of this study was to evaluate the effects of adding different concentrations of edible bird’s nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus^® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus^® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin^® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.     

Effect of semen collection method on sperm motility of gray wolves (Canis lupus) and domestic dogs (C. l. familiaris)

Genetic management of Mexican gray wolves includes semen banking, but due to the small number of animals in the population and handling restrictions, improvements in semen collection and cryopreservation rely on results from studies of domestic dogs. Semen collection from wolves requires anesthesia and electroejaculation, which introduce potentially important variables into species comparisons, as dog semen is typically collected manually from conscious animals. To investigate possible effects of collection method on semen quality, we compared semen collection by the traditional manual method and by electroejaculation (EE) in a group of dogs (n = 5) to collection by EE only in wolves (n = 7). Samples were divided into two aliquots: neat or diluted in Tris/egg yolk extender, with motility evaluated at intervals up to 24 h. There were no differences (P > 0.10) in sperm motility in either neat or extended samples at 24 h from EE dogs and wolves, although motility of the wolf neat samples declined more rapidly (P < 0.05). However, there were differences (P < 0.01) between EE and manually collected dog semen in motility at 24 h, in both the neat and extended samples. Therefore, general motility patterns of dog and wolf semen collected by EE were similar, especially when diluted with a Tris/egg yolk extender, but sperm collected from dogs by EE did not maintain motility as long as manually collected samples, perhaps related to the longer exposure of EE samples to more prostate fluid.     

Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa

New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H₂O₂ and O₂ production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24h, 48h and 72h.Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24h, 48h and 72h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.     

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.     

Effect of dialysis on quality characteristics of turkey semen during liquid storage

Low molecular weight substances such as zinc and peroxides are present in seminal plasma and are responsible for deleterious effects in stored semen. On the contrary, molecules larger than 50 kDa are beneficial to in-vitro storage of spermatozoa. Since the effects of different seminal plasma fractions in turkey semen are not completely known, the purpose of the study was to determine the effects of turkey semen dialysis with a 12-14 kDa cut-off on viability, hypo-osmotic membrane integrity, or sperm motility of turkey spermatozoa stored up to 48 h at 5 degrees C. Twelve pools of semen, each pool originating from four toms, were used. Each pool was divided into two aliquots, one of which was dialyzed while the other represented the control. Each semen aliquot was evaluated for sperm viability, membrane integrity and motility after 6, 24 and 48 h of in-vitro storage. Cold storage of turkey semen for 48 h significantly worsened (P<0.01) sperm viability, hypo-osmotic membrane integrity, and sperm motility index of both control and dialyzed samples. After 24 and 48 h sperm viability, membrane integrity and sperm motility index were better (P<0.01) in dialyzed semen compared to the control.     

The spermatozoon of the African catfish: fine structure, motility, viability and its behaviour in seminal vesicle secretion

The spermatozoon of the African catfish Clarias gariepinus is a simple organized aquasperm although it reveals very unique characteristics: the cytoplasmic channel is lacking, the mitochondria form a complex structure and the arrangement of the centriolar complex is species specific. Semen has high initial motility rates ( c. 70–90%) and swimming velocities ( c. 120–140 μm s⁻¹), the main swimming type is linear. Motility duration in water is 30 s and is prolonged only to 40 s in NaCl solutions or more complex bu ered motility activating saline solutions. A pH between 7.0 and 9.0 has no e ect on the sperm motility parameters. Motility is completely and reversibly suppressed in electrolyte and non-electrolyte solutions with an osmolality of 200 mosmol kg⁻¹. During immotile storage the sperm viability is influenced by the osmolality and the potassium levels of the storage medium, by the temperature and by the dilution. At optimal conditions (bu ered sperm motility inhibiting saline solution: 150 mmol l⁻¹ NaCl, 2.5 mmol l⁻¹ KCl, 1 mmol l⁻¹ CaCl₂, 1 mmol l⁻¹ MgSO₄, 20 mmol l⁻¹ Tris solution, pH 8.5; dilution rate 1: 5; storage temperature, 4°C) sperm viability persists for >7 days. High viscosity of the pure seminal vesicle secretion completely inhibits the sperm motility. When the seminal vesicle secretion is diluted in water the viscosity decreases and the motility suppressing e ect is neutralized. When semen is mixed with seminal vesicle secretion the sperm viability decreases to zero within 10 min.